The biochemistry and physiology of RNA splicing have become a significant area of research recently. We will continue our analysis of the pre-tRNA splicing pathway in the yeast, Saccharomyces cerevisiae. The biochemical mechanism of each of the two enzymes, the cleavage endonuclease and the RNA kinase-ligase, that have been identified and partially purified on the basis of their participation in the splicing reaction in vitro, will be investigated further. We expect to focus on the cleavage enzyme, as its specificity determines the location and precision of splicing. The requirements and reaction intermediates of the cleavage activity will be investigated with respect to the fidelity and rate of splicing. Assays will be developed to identify two additional factors which seem to be required to reconstitute the entire pre-tRNA splicing reaction. The cleavage enzyme is a membrane-bound protein, and there is reason to believe that it is part of the nuclear-membrane. That hypothesis will of the enzyme. Mutants of yeast defective for pre-tRNA splicing will be analyzed to determine whether either the activity or lthe subcellular localization of these enzymes is aberrant. We will look in protein-processing mutants, isolated on the basis of defects in secretion, for altered localization. One of the in vitro requirements for accurate cleavag is the addition of a polyamine cation. This prompts us to study small RNA metabolism and splicing enzymes in mutants which are unable to synthesize polyamines. Finally, we expect to develop an RNA-filter colony-replacia technique and employ it to identify mutants which accumulate pre-tRNAs at the non-permissive temperature. This will be complemented by studies of suppressors of one known mutant of yeast which accumulates pre-tRNAs.